CN109900843B - Method for simultaneously detecting 7 conjugated estrogens in livestock and poultry manure by combining microwave extraction-solid phase extraction pretreatment with liquid chromatography-mass spectrometry technology - Google Patents
Method for simultaneously detecting 7 conjugated estrogens in livestock and poultry manure by combining microwave extraction-solid phase extraction pretreatment with liquid chromatography-mass spectrometry technology Download PDFInfo
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Abstract
The invention relates to a microwave extraction-solid phase extraction pretreatment combined liquid chromatography-mass spectrometry technologyA method for detecting 7 conjugated estrogens in animal and fowl feces. The method comprises the following steps: (1) pretreating fecal samples, respectively adding 17 beta-estradiol D 3 3-beta-D-glucuronide, 17 beta-sodium estradiol sulfate-D 4 These two internal standards indicate recovery; performing microwave extraction with an extraction solvent, filtering, collecting the extract, diluting with water, and adjusting pH; (2) enriching and purifying target estrogen by solid-phase extraction; (3) an internal standard method is adopted, and 7 conjugated estrogens in the livestock and poultry manure are quantitatively detected by a liquid chromatograph-mass spectrometer. The method optimizes the extraction method, the enrichment purification condition and the liquid quality detection condition, simultaneously selects a proper internal standard substance, can simultaneously detect the residual conditions of 7 combined estrogens in the livestock and poultry manure at one time, and has the advantages of high recovery rate, high sensitivity, high stability, low detection limit, more real and reliable detection result and the like.
Description
Technical Field
The invention belongs to the technical field of organic pollutant residue detection, relates to a method for detecting conjugated estrogens in livestock and poultry manure, and particularly relates to a method for simultaneously detecting the contents of 7 conjugated estrogens in livestock and poultry manure by combining microwave extraction-solid phase extraction pretreatment with a liquid chromatography tandem mass spectrometry technology.
Technical Field
With the rapid development of livestock and poultry breeding towards modernization, scale and intensification, the pollution of livestock and poultry breeding is increasingly serious, and the estrogen entering the environment through livestock and poultry manure exceeds the environmental capacity. Endogenous steroid estrogen enters the environment from excrement and urine of livestock and poultry farms, and trace endogenous steroid estrogen can strongly interfere with endocrine systems of human beings and other animals, and change intracellular signal peptide processes of organisms in development and adult stages, so that development, behavior and reproduction problems are caused. The livestock and poultry manure matrix is a complex environmental sample, and effective sample extraction and purification are the prerequisites for accurately determining the estrogen content in the livestock and poultry manure matrix. Therefore, an effective extraction and purification method is particularly important for determining the content of estrogen in the livestock and poultry manure.
The extraction method commonly used in the sample pretreatment mainly comprises a Soxhlet extraction method, an accelerated solvent extraction method, an ultrasonic extraction method, a microwave extraction method and the like. The microwave extraction method directly heats the solvent and the sample in the container, the sample and the solvent can be heated to a high temperature in a short time, the heating rate is improved, the solvent extraction efficiency is enhanced, and the method has the advantages of short time, reagent saving, high recovery rate and the like, and is particularly suitable for complex matrixes such as feces and the like. Meanwhile, the selection of the extracting agent has direct influence on the recovery rate. The ideal pretreatment method of the environmental sample does not destroy the form of the component to be detected, reduces pollution and can effectively separate the component to be detected from the matrix. The detection methods commonly used at present mainly comprise high performance liquid chromatography (HLPC) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). HPLC-MS/MS has both strong separation capability of HLPC, high sensitivity of MS and extremely strong qualitative identification capability, is one of the most effective means for detecting estrogen residues in complex matrix binding state in the current environment, and has the advantages of wide analysis range, strong separation capability, reliable qualitative analysis result, low detection limit, quick analysis time, high automation degree and the like. The components of the complex mixture can be accurately, qualitatively and quantitatively determined.
At present, no complete and reliable detection method for separation, enrichment and co-detection of the conjugated estrogens in the animal and poultry feces exists, so that a method which has stable detection result, small interference, rapidness, high efficiency, sensitivity and accuracy and can simultaneously detect a plurality of conjugated estrogens in the animal and poultry feces is urgently needed, and the complete and reliable detection method comprises separation, enrichment and co-detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for simultaneously determining 7 conjugated estrogens in livestock and poultry manure with stable detection result, small interference, rapidness and high efficiency, namely a method for simultaneously detecting 7 conjugated estrogens in livestock and poultry manure by combining a microwave extraction-solid phase extraction pretreatment and a liquid chromatography-mass spectrometry technology.
The technical concept of the invention is as follows:
the method adopts microwave extraction combined with solid phase extraction as the pretreatment of the livestock and poultry manure sample, and adopts high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) as a detector to simultaneously determine the content of 7 conjugated estrogens in the sample. Selecting 7 kinds of conjugated estrogens estrone 3- (BETA-D-glucuronide) sodium salt (E1-3G), 17 BETA-estradiol 17-potassium sulfate salt (E2-S-K), BETA-estradiol 3-sodium sulfate salt (E2-3S), 17 BETA-estradiol 3- (BETA-D-glucuronide) sodium salt (E2-3G), estriol-3-O-BETA-D-glucuronide sodium salt (E3-3G), estrone 3-sodium sulfate (E1-3S) and 17 BETA-estradiol 17-BETA-D-glucuronide (E2-G) which are common in livestock and poultry feces, and adopting a one-factor test to select optimal conditions to obtain the maximum extraction rate of conjugated estrogens under the optimal conditions.
The purpose of the invention is realized by the following technical scheme:
a method for simultaneously detecting 7 conjugated estrogens in livestock and poultry manure by combining microwave extraction-solid phase extraction pretreatment with a liquid chromatography-mass spectrometry technology comprises the following steps:
(1) extraction of conjugated estrogens: microwave extraction
Weighing a livestock and poultry manure sample which is subjected to freeze drying, crushing and sieving, adding an internal standard substance indicating the recovery rate, adding an extraction solvent, performing microwave extraction and filtration, collecting an extracting solution, diluting with ultrapure water, and adjusting the pH value;
the internal standard substance for indicating the recovery rate is 17 beta-estradiol D 3 3-beta-D-glucuronide (E2-3G-D) 3 ) 17 beta-estradiol sodium sulfate D 4 (E2-3S-D 4 )；
(2) Enrichment and purification: solid Phase Extraction (SPE)
Activating a solid phase extraction column by using equal volumes of methyl tert-butyl ether, methanol and ultrapure water in sequence, enabling the sample treated in the step (1) to pass through the column, and controlling the flow rate of the sample passing through the column; after the enrichment is finished, leaching the solid phase extraction column by using leacheate, and drying the solid phase extraction column in vacuum; eluting the target substance with methanol of a certain volume, purifying the eluent with an amino column activated by methanol, and blow-drying with a nitrogen blowing instrument to obtain residue; dissolving the residue with an organic solvent with a certain concentration, fixing the volume, performing ultrasonic treatment, filtering, transferring to a sample injection bottle, and testing;
(3) high performance liquid chromatography tandem mass spectrometry for determining content of 7 conjugated estrogens in livestock and poultry manure
And (3) quantitatively detecting the content of 7 conjugated estrogens enriched from the livestock and poultry manure in the sample to be detected in the sample injection bottle obtained by the treatment of the step (2) by adopting an internal standard method and using a high performance liquid chromatography tandem mass spectrometer. The 7 conjugated estrogens are estrone 3- (BETA-D-glucuronide) sodium salt (E1-3G), 17 β -estradiol 17-potassium sulfate salt (E2-S-K), BETA-estradiol 3-sodium sulfate salt (E2-3S), 17 β -estradiol 3- (β -D-glucuronide) sodium salt (E2-3G), estriol-3-O- β -D-glucuronide sodium salt (E3-3G), estrone 3-sodium sulfate (E1-3S), and 17 β -estradiol 17- β -D-glucuronide (E2-G), respectively.
The further technical scheme is as follows:
in the step (1), 100ng of 17 beta-estradiol D is respectively added before microwave extraction 3 3-beta-D-glucuronide (E2-3G-D) 3 ) 17 beta-estradiol sodium sulfate D 4 (E2-3S-D 4 ) Both internal standards indicate recovery.
In the step (1), the extraction solvent is methanol, a citric acid buffer solution with pH value of 5 (V: 1), namely a mixed solution of methanol and a citric acid buffer solution with pH value of 5, wherein the volume ratio of the methanol to the citric acid buffer solution with pH value of 5 is 1: 1; and (3) extracting by using an CEM MARS CLASSIC type microwave extractor to extract the conjugated estrogen in the excrement.
In the step (1), the screen used for sieving the excrement sample is a screen with the aperture of 0.25 mm. The glass fiber filter used for filtering the extract after the microwave extraction is not particularly limited, and may have a pore size of 0.45 μm, 0.47 μm or 0.7 μm, for example, for the purpose of removing the feces remaining in the extract and preventing the subsequent influence on the enrichment and detection.
In the step (1), the pH value of the diluted sample is adjusted to 3-4, and no specific numerical value is required.
In the step (2), the solid phase extraction column used for enrichment and purification is an Oasis HLB small column of Waters company, which is a hydrophilic-lipophilic polymer packed column, and the adsorbent (packing) is a macroporous copolymer polymerized by two monomers of lipophilic divinylbenzene and hydrophilic N-vinyl pyrrolidone according to a certain proportion, and is a general adsorbent for acidic, neutral and alkaline compounds.
In the step (2), the amino column used for purification is NH of Waters 2 Cartridges with a polar stationary phase and a weak anion exchanger, which can be retained by weak anion exchange (aqueous solution) or polar adsorption (non-polar organic solution), i.e. NH of the amino column 2 The (aminopropyl) bonded phase has strong polar adsorption in a nonpolar organic solvent and has weak anion exchange retention.
In the step (2), the flow rate of the sample through the solid phase extraction column is not particularly limited. In order to ensure sufficient adsorption of the target substance on the solid phase extraction column, the sample flow rate (i.e., the flow rate of the sample through the column) should preferably not exceed 5 mL/min.
In the step (2), the dosage of methyl tert-butyl ether, methanol and ultrapure water used for activating the solid-phase extraction column is 5-10 mL; the dosage of the methanol used for activating the amino column is 3-6 mL; the eluent used for leaching the solid phase extraction column is low-concentration methanol aqueous solution, ultrapure water and 5-10mL of ammonia water-methanol-water respectively with the volume concentration ratio of 2:10: 88; vacuum drying for 20-30 min; the flow rate when the solid phase extraction column is eluted is lower than 10 mL/min;
in the step (2), the low-concentration methanol used for leaching the solid-phase extraction column is methanol-water solution with volume concentration less than 10%; dissolving the residue with organic solvent, diluting to desired volume, and performing ultrasonic treatment for 5-10 min; filter to a brown injection vial with a needle filter; the needle filter adopts a polytetrafluoroethylene needle filter head with the pore diameter of less than 0.22 mu m.
Among these, the ratio of methanol to water in the low-concentration methanol used for eluting the solid-phase extraction column is not particularly limited. The objective is to wash away unwanted impurities from the solid phase extraction column, but to prevent the possibility of simultaneous elution of the target substances, the aqueous solution used for the washing should not have a methanol content of more than 10%.
In the step (2), the organic solvent used for dissolving the residual substances after nitrogen blowing is not particularly limited, and methanol or acetonitrile, or a methanol-water solution with a volume concentration of 70-80%, or an acetonitrile-water solution with a volume concentration of 70-80% can be selected, such as: methanol-water solution with the volume ratio of methanol to water being 7:3, acetonitrile-water solution with the volume ratio of acetonitrile to water being 8:2, and the like.
In the step (3), an AB5500Q-trap high performance liquid chromatography tandem mass spectrometer of American AB company is selected, and Shimadzu 30A liquid chromatography is matched to detect 7 target binding state estrogen substances; the liquid chromatography column used was Shim-Pack XR-ODSII C18 column type 75mm × 2mm, 1.0 μm.
In the step (3), the liquid chromatography separation parameters are as follows: the column temperature of the chromatographic column is 40 ℃; the sample size is 4 mu L; the temperature of a sample injection chamber is 10 ℃; flow rate of mobile phase 0.3 mL/min -1 (ii) a The mobile phase A is an ammonia water-water solution with the volume concentration of 0.1 percent, wherein the water is Milli-Q ultrapure water; the mobile phase B is acetonitrile; the gradient elution procedure was: 0-2min, 10% B; 2.0-2.2min, 10% B-50% B; 2.2-3.5min, 50% B; 3.5-3.8min, 50% B-97% B; 3.8-5.5min, 97% B; 5.5-5.6min, 97% B-10% B; 10min, 10% B.
In the step (3), the electrospray ion source negative ion mode ESI-, triple quadrupole mass analyzer, scanning mode: detecting in a multi-reaction monitoring mode MRM; the ion source temperature is 550 ℃, the ionization voltage is-4500V, the air curtain gas CUR is 35psi, the spray gas GS1 is 50psi, the auxiliary heating gas GS2 is 50psi, the collision gas CAD is Medium, and the collision gas is high-purity nitrogen.
The invention has the beneficial effects that:
the detection method provided by the invention adopts a microwave extraction-solid phase extraction-high performance liquid chromatography tandem mass spectrometry technology, and can realize rapid, efficient, sensitive and accurate simultaneous detection of 7 binding-state estrogens in livestock and poultry manure by optimizing an extraction method, an enrichment purification condition and a liquid quality detection condition, so that the detection efficiency is greatly improved, the detection cost is reduced, and the detection result is stable and has small interference.
Compared with the prior art, the invention has the following advantages:
1. the invention adopts a microwave extraction method to extract the conjugated estrogen in the excrement. Compared with the oscillation extraction, the method can effectively improve the extraction efficiency, reduce the dosage of the extracting agent, reduce the extraction time, improve the recovery rate and achieve the aim of extracting the target substance.
2. The invention selects the Oasis HLB solid-phase extraction column to enrich and purify 7 conjugated estrogens, has high and stable recovery rate, strong selectivity and large adsorption capacity, and achieves the aim of effectively separating and enriching the target substances.
3. NH of Waters corporation 2 The Cartidges column further purifies the 7 conjugated estrogens to remove pigments, so that the sample inlet and the chromatographic column can be well protected from being polluted when a sample to be tested is tested on a loading machine, and the service life is prolonged.
4. The method adopts an internal standard curve method to quantify the conjugated estrogen, has good linear relation of the measuring result and small relative deviation, and improves the precision of analysis.
5. The invention adopts the high performance liquid chromatography tandem mass spectrometer for quantitative analysis and detection, and has high sensitivity. In addition, the tandem triple quadrupole mass spectrometer carries out qualitative analysis according to characteristic fragment ions generated by the collision of corresponding parent ions, has strong selectivity, and eliminates the interference of other impurity signals in a sample.
6. According to the method, the internal standard substance indicating the recovery rate is added before microwave extraction, so that the loss of the combined estrogen in the whole pretreatment process can be more accurately represented, meanwhile, the loss of different types of estrogens in different degrees in the pretreatment process is considered, and the same internal standard substance is selected for estrogens with similar structures to indicate the recovery rate; in the invention, 17 beta-estradiol D is respectively selected 3 3-beta-D-glucuronide (E2-3G-D) 3 ) As estrone 3- (BETA-D-glucuronide) sodium salt (E1-3G), 17 β -estradiol 3- (β -D-glucuronide) sodium salt (E2-3G), 17 β -estradiol 17- β -D-glucuronide (E2-G), estriol-3-O- β -D-glucuronide sodium salt (E3-3G) The internal standard substance of (1) is 17 beta-sodium estradiol sulfate-D 4 (E2-3S-D 4 ) As the internal standard substances of estrone 3-sodium sulfate (E1-3S), BETA-estradiol 3-sodium sulfate (E2-3S) and 17-estradiol 17-potassium sulfate (E2-S-K), the final detection result is more real and reliable due to the proper selection of the internal standard substances.
7. The invention has the advantages of small usage amount of organic reagent and environmental protection. 7 conjugated estrogens in excrement can be detected by one-time sample injection, the detection process consumes less time, and the cost of manpower and material resources is saved.
Drawings
FIG. 1 is a graph of normalized recovery of 7 conjugated estrogens in cow dung using three different extractants.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
The following provides a specific implementation mode and content of a method for simultaneously detecting 7 conjugated estrogens in livestock and poultry manure by combining microwave extraction-solid phase extraction pretreatment with a liquid chromatography-mass spectrometry technology.
Example 1: standard recovery rate experiment
The influence of the microwave extraction-solid phase extraction pretreatment combined liquid chromatography-mass spectrometry detection method on the recovery rate of 7 combined estrogen in the livestock and poultry manure is examined by adopting an internal standard method. The standard recovery rate experiment takes cow dung as an example: 100ng and 200ng of mixed standard solutions (mixed standard solutions of 7 conjugated estrogens) were added to feces, respectively. The method is implemented by the following steps:
step one, extraction of livestock and poultry manure
The collected feces were freeze-dried, ground and sieved with a 0.25mm mesh. Two groups of stool samples for comparison test were set, one group was a standard sample group, the other group was a blank sample group, and each group was set in parallel. 1.00g of each fecal sample was accurately weighed into a 100mL beaker. To the standard sample set, 100ng and 200ng of the mixed standard solution of conjugated estrogens (i.e., the mixed standard solution of 7 conjugated estrogens) were added, respectively, to give final concentrations of conjugated estrogens 100. mu.g/kg and 200. mu.g/kg in feces. The blank group did not add any conjugated estrogens.
100ng of 17 beta-estradiol D was added to each stool sample 3 3-beta-D-glucuronide (E2-3G-D) 3 ) 17 beta-estradiol sodium sulfate D 4 (E2-3S-D 4 ) The two internal standards indicating recovery rates were thoroughly mixed with the fecal sample and dried in a fume hood for 30 min.
Transferring the excrement sample in the beaker to a microwave extraction tank, adding 50mL of methanol and citric acid buffer solution (V: 1) with pH value of 5 as an extraction solvent, performing microwave extraction at the extraction temperature of 95 ℃ and the extraction power of 800W, wherein the extraction time gradient is set as follows: the temperature is increased for 10min, maintained for 60min and reduced for 20 min. After the extraction, the extract was filtered, and the extract was collected, diluted to 500mL with distilled water, and the pH was adjusted to 3.
Step two, pretreatment of Solid Phase Extraction (SPE): enrichment purification process
Using a Reeko Foctor Plus full-automatic solid phase extractor, an Oasis HLB solid phase extraction column, NH 2 And (4) enriching and purifying the conjugated estrogen by using a cartidges column. The solid phase extraction procedure was as follows: for Oasis HLB solid phase extraction column, methyl tert-butyl ether activation (5mL/min,6mL), methanol activation (5mL/min,6mL), ultrapure water H 2 O activation (5mL/min,6mL), bulk loading (5mL/min, 500 mL); after the sample loading is finished, sequentially eluting with 8% methanol-water solution (5mL/min,6mL), 3-pH water (5mL/min,6mL) and 2:10:88 ammonia-methanol-water (5mL/min,6 mL); then, the extraction column is dried for 5min under a vacuum state; methanol elution (5mL/min,10 mL); to NH 2 Performing a cartidges column, activating with methanol (5mL/min,6mL), loading (4mL/min, 10mL), collecting the loading solution, and drying with nitrogen gas to obtain a residue; dissolving with 80% methanol, diluting to 1mL, performing ultrasonic treatment for 5min, filtering with 0.22 μm PTFE needle filter, transferring the solution sample to brown sampling bottle, and testing.
Conjugated estrogens belong to the class of steroids, either apolar or apolar substances. According to the invention, through experiments, two conditions that the solid-phase extraction column is activated by methanol and ultrapure water in sequence and the solid-phase extraction column is activated by methyl tert-butyl ether, methanol and ultrapure water in sequence are compared, and the fact that the solid-phase extraction column has higher recovery rate and repetition rate on conjugated estrogen substances is found. In the invention, three conditions that the solid phase extraction column is respectively leached by methanol-water solutions with volume concentrations of 12%, 8% and 5% are compared through experiments, and the fact that the solid phase extraction column is leached by the methanol-water solution with the volume concentration of 8% has high relative recovery rate and can remove part of impurities is found, and then the impurities dissolved in the acidic solution and the alkaline solution are respectively leached by an ultrapure water solution with pH being 3 and an ammonia-methanol-water solution with the volume concentration ratio of 2:10:88 (namely V ammonia/V methanol/V ultrapure water being 2/10/88), so that the impurities such as inorganic salts, macromolecular substances, pigments and the like in a sample can be better removed.
The proportion of methanol in the methanol-water solution of a certain proportion used for washing the solid-phase extraction column is not particularly limited, and the purpose is to wash off impurity components remaining on the solid-phase extraction column, but in order to prevent the possibility of simultaneous washing of the target substance, the proportion of the volume concentration of methanol in the water solution used for washing is preferably not more than 10%.
Step three, determining the content of 7 conjugated estrogens by liquid chromatography tandem mass spectrometry
And (3) quantitatively detecting the content (concentration) of the 7 conjugated estrogens in the livestock and poultry manure sample in the sampling bottle on a liquid chromatogram-tandem mass spectrometer by adopting an internal standard method. The detection conditions were as follows:
an AB5500Q-trap high performance liquid chromatography tandem mass spectrometer of American AB company is selected, and Shimadzu 30A liquid chromatography is matched to detect 7 target binding state estrogen substances; the liquid chromatography column used was Shim-Pack XR-ODSII C18 column type 75mm × 2mm, 1.0 μm. .
The liquid chromatography separation parameters were: the column temperature of the chromatographic column is 40 ℃; the sample size is 4 mu L; the temperature of a sample injection chamber is 10 ℃; flow rate of mobile phase 0.3 mL/min -1 (ii) a The mobile phase A is an ammonia water-water solution with the volume concentration of 0.1 percent, wherein the water is Milli-Q ultrapure water; the mobile phase B is acetonitrile; the gradient elution procedure was: 0-2min, 10% B; 2.0-2.2min, 10%B-50％B；2.2-3.5min，50％B；3.5-3.8min，50％B-97％B；3.8-5.5min，97％B；5.5-5.6min，97％B-10％B；10min，10％B。
The mass spectrum detection conditions are as follows: electrospray ion source anion mode ESI-, triple quadrupole mass analyzer, scanning mode: detecting in a multi-reaction monitoring mode MRM; the ion source temperature is 550 ℃, the ionization voltage is-4500V, the air curtain gas CUR is 35psi, the spray gas GS1 is 50psi, the auxiliary heating gas GS2 is 50psi, the collision gas CAD is Medium, and the collision gas is high-purity nitrogen.
Considering that different conjugated estrogens have different structures and properties and different losses in the pretreatment process, 17 beta-estradiol D is selected respectively 3 3-beta-D-glucuronide (E2-3G-D) 3 ) 17 BETA-sodium estradiol sulfate-D-glucuronide (E1-3G), 17 BETA-estradiol 3- (BETA-D-glucuronide) sodium salt (E2-3G), 17 BETA-estradiol 17-BETA-D-glucuronide (E2-G), and estriol-3-O-BETA-D-glucuronide sodium salt (E3-3G) as internal standard substances 4 (E2-3S-D 4 ) As internal standard substances of estrone 3-sodium sulfate (E1-3S), BETA-estradiol 3-sodium sulfate (E2-3S), and 17 β -estradiol 17-potassium sulfate (E2-S-K).
And (3) calculating the recovery rate:
and (3) pretreating by adopting a pretreatment method from the first step to the second step, quantitatively detecting by adopting a method from the third step, and calculating the standard adding recovery rate of the cow dung sample (the final concentration of the standard adding is 100 mug/kg), wherein the calculation results are shown in table 1.
The normalized recovery (RE%) is calculated by the formula:
wherein, RE: recovery rate of standard addition,%;
c0: the concentration of the mixed standard solution is ng/mL;
c1: the detection concentration of the excrement sample without the mixed standard solution is ng/mL;
c2: adding the detection concentration of the excrement sample of the mixed standard solution, wherein the detection concentration is ng/mL;
v0: volume of mixed standard solution, mL;
v1: before a machine, the volume of the excrement sample which is not added with the mixed standard solution is determined to be mL;
v2: and (4) adding the excrement sample mixed with the standard solution into the machine, and fixing the volume to mL.
It can be seen that the recovery of the 7 conjugated estrogens by the method of the present invention is between 70.5% and 115.9%, and the relative standard deviation is less than 5%. The difference of the standard recovery rate indicates that the matrix interference exists; the influence caused by matrix interference can be reduced to a certain extent by calculating the recovery rate of the added standard by adding the internal standard substance and correcting the detection result by using the recovery rate of the added standard.
TABLE 1 measured concentration of conjugated estrogens in cow dung samples and spiked recovery for different spiked concentrations
The standardized recovery rates of 7 conjugated estrogens in livestock and poultry feces were determined by testing 3 different extraction solvents (methanol: citric acid buffer solution with pH 5 (V: 1), namely methanol: citric acid buffer solution with pH 5 in a volume ratio of 1:1, EDTA-Mcllvaine buffer solution (V: 1), namely a mixed solution of methanol and EDTA-Mcllvaine buffer solution with a volume ratio of 1:1, and acetone (V: 1:3), namely a mixed solution of methanol and acetone with a volume ratio of 1:3), respectively, as shown in fig. 1. As can be seen from fig. 1, the highest recovery rate and the best effect were obtained by using methanol, citric acid buffer solution (V: 1) with pH 5 as the extraction solvent. Therefore, in the actual detection of conjugated estrogens in animal and poultry feces, methanol, citric acid buffer solution with pH 5 (V: 1) can be selected as extraction solvent.
Examples 2 to 3: determination of estrogen content in actual stool samples
Determination of the concentration of 7 conjugated estrogens in different fecal samples
The method comprises the steps of collecting cow dung of three different types of cows in a certain cow farm in Shanghai, performing sample pretreatment by adopting the method (microwave extraction and liquid-liquid extraction) in the first step, performing enrichment and purification pretreatment by adopting the solid phase extraction method in the second step, and detecting and analyzing the actual concentration of the sample by adopting the liquid chromatography-mass spectrometry detection method in the third step, so as to examine the applicability of the method to different types of manure samples.
The method is implemented by the following steps:
(1) extraction of conjugated estrogens: microwave extraction
The three collected cow dung are respectively freeze-dried, crushed and sieved by a 0.25mm sieve. Accurately weigh 1.00g of each stool into a 100mL microwave extraction tube. 100ng of 17 beta-estradiol D was added to each stool 3 3-beta-D-glucuronide (E2-3G-D) 3 ) 17 beta-estradiol sodium sulfate D 4 (E2-3S-D 4 ) The two internal standards indicating recovery rates were thoroughly mixed with the fecal sample and then dried in a fume hood for 30 min.
Then 50mL of methanol and citric acid buffer solution with pH value of 5 (V: 1) are added as an extraction solvent for microwave extraction, the extraction temperature is 95 ℃, the extraction power is 800W, and the extraction time gradient is set as follows: the temperature is increased for 10min, maintained for 60min and reduced for 20 min. After the extraction, the extract was filtered, and the extract was collected, diluted to 500mL with distilled water, and the pH was adjusted to 3.
(2) Solid Phase Extraction (SPE): enrichment purification process
Using a Reeko Foctor Plus full-automatic solid phase extractor, an Oasis HLB solid phase extraction column, NH 2 And (4) enriching and purifying the conjugated estrogen by using a cartidges column. The solid phase extraction procedure was as follows: for Oasis HLB solid phase extraction column, methyl tert-butyl ether activation (5mL/min,6mL), methanol activation (5mL/min,6mL), H 2 O activation (5mL/min,6mL), bulk loading (5mL/min, 500 mL); after the sample loading is finished, sequentially eluting with 8% methanol-water solution (5mL/min,6mL), 3-pH water (5mL/min,6mL) and 2:10:88 ammonia-methanol-water (5mL/min,6 mL); then, the extraction column is dried for 5min under a vacuum state; methanol elution(5mL/min,10 mL); to NH 2 Performing a cartidges column, activating with methanol (5mL/min,6mL), loading (4mL/min, 10mL), collecting the loading solution, and drying with nitrogen gas to obtain a residue; dissolving with 80% methanol, diluting to 1mL, performing ultrasonic treatment for 5min, filtering with 0.22 μm PTFE needle filter, transferring the solution sample to brown sampling bottle, and testing.
(3) High performance liquid chromatography tandem mass spectrometer for quantitatively detecting content of 7 conjugated estrogens in feces
Quantitatively detecting the content (concentration) of 7 conjugated estrogens in the livestock and poultry manure sample in the sampling bottle on a liquid chromatogram-tandem mass spectrometer by adopting an internal standard method; the 7 conjugated estrogens are estrone 3- (BETA-D-glucuronide) sodium salt (E1-3G), 17 β -estradiol 17-potassium sulfate salt (E2-S-K), BETA-estradiol 3-sodium sulfate salt (E2-3S), 17 β -estradiol 3- (β -D-glucuronide) sodium salt (E2-3G), estriol-3-O- β -D-glucuronide sodium salt (E3-3G), and estrone 3-sodium sulfate (E1-3S).
The detection conditions were as follows:
an AB5500Q-trap high performance liquid chromatography tandem mass spectrometer of American AB company is selected, and is matched with Shimadzu 30A liquid chromatography to detect 7 target binding state estrogen substances; the liquid chromatography column used was Shim-Pack XR-ODSII C18 column type 75mm × 2mm, 1.0 μm.
The liquid chromatography separation parameters were: the column temperature of the chromatographic column is 40 ℃; the sample volume is 4 mu L; the temperature of a sample injection chamber is 10 ℃; flow rate of mobile phase 0.3 mL/min -1 (ii) a The mobile phase A is an ammonia water-water solution with the volume concentration of 0.1 percent, wherein the water is Milli-Q ultrapure water; the mobile phase B is acetonitrile; the gradient elution procedure was: 0-2min, 10% B; 2.0-2.2min, 10% B-50% B; 2.2-3.5min, 50% B; 3.5-3.8min, 50% B-97% B; 3.8-5.5min, 97% B; 5.5-5.6min, 97% B-10% B; 10min, 10% B.
The mass spectrum detection conditions are as follows: electrospray ion source anion mode ESI-, triple quadrupole mass analyzer, scanning mode: detecting in a multi-reaction monitoring mode MRM; the ion source temperature is 550 ℃, the ionization voltage is-4500V, the air curtain gas CUR is 35psi, the spray gas GS1 is 50psi, the auxiliary heating gas GS2 is 50psi, the collision gas CAD is Medium, and the collision gas is high-purity nitrogen.
The actual detection results show that the content of 7 conjugated estrogen substances in three types of excrement is shown in the table 2. Experimental results show that the method can be applied to determination of the content of the conjugated estrogen in the livestock and poultry manure and has good applicability.
TABLE 2 content determination of 7 conjugated estrogens in three excreta
Note: data are mean ± standard deviation (n ═ 3).
Claims (7)
1. A method for simultaneously detecting 7 conjugated estrogens in animal manure by combining microwave extraction-solid phase extraction pretreatment and a liquid chromatography-mass spectrometry technology is characterized by comprising the following steps:
(1) extraction of conjugated estrogens: microwave extraction
Weighing a livestock and poultry manure sample subjected to freeze drying, crushing and sieving, adding an internal standard substance indicating the recovery rate, adding an extraction solvent, performing microwave extraction and filtration, collecting an extracting solution, diluting with ultrapure water, and adjusting the pH value;
wherein the extraction solvent is a mixed solution of methanol and a citric acid buffer solution with pH value of 5, wherein the volume ratio of the methanol to the citric acid buffer solution with pH value of 5 is 1: 1; extracting by using an CEM MARS CLASSIC type microwave extractor to extract the conjugated estrogen in the excrement; the screen used for filtering the livestock and poultry manure sample is a screen with the aperture of 0.25 mm; the aperture of a glass fiber filter membrane used for filtering the extracting solution after microwave extraction is 0.45 μm, 0.47 μm or 0.7 μm; adjusting the pH value of the diluted sample to 3-4; the internal standard substance for indicating the recovery rate is 17 beta-estradiol D 3 3-beta-D-glucuronide, 17 beta-sodium estradiol sulfate-D 4 ；
(2) Enrichment and purification: solid phase extraction
Activating a solid phase extraction column by using equal volumes of methyl tert-butyl ether, methanol and ultrapure water in sequence, enabling the sample treated in the step (1) to pass through the column, and controlling the flow rate of the sample passing through the column; after enrichment, leaching the solid-phase extraction column by using leacheate, and drying the solid-phase extraction column in vacuum; eluting the target substance with methanol of a certain volume, purifying the eluent with an amino column activated by methanol, and blow-drying with a nitrogen blowing instrument to obtain residue; dissolving the residue with an organic solvent with a certain concentration, fixing the volume, performing ultrasonic treatment, filtering, transferring to a sample injection bottle, and testing;
wherein, the solid phase extraction column used for enrichment is an Oasis HLB column of Waters company, which is a hydrophilic-lipophilic polymer packed column, the adsorbent is a macroporous copolymer polymerized by two monomers of lipophilic divinylbenzene and hydrophilic N-vinyl pyrrolidone according to a certain proportion, and the adsorbent is a general adsorbent used for acidic, neutral and alkaline compounds; the amino column used for purification was Waters NH 2 A cartidges column, which has a polar stationary phase and a weak anion exchanger and can achieve retention by weak anion exchange or polar adsorption; the flow rate of the sample passing through the column is not more than 5 mL/min; the flow rate of the activated solid phase extraction column is not more than 5 mL/min;
wherein the dosage of methyl tert-butyl ether, methanol and ultrapure water used for activating the solid-phase extraction column is 5-10 mL; the dosage of the methanol used for activating the amino column is 3-6 mL; the eluent used for leaching the solid phase extraction column is low-concentration methanol aqueous solution, ultrapure water and 5-10mL of ammonia water-methanol-water respectively with the volume concentration ratio of 2:10: 88; vacuum drying for 20-30 min; the flow rate when eluting the solid phase extraction column is lower than 10 mL/min;
(3) high performance liquid chromatography tandem mass spectrometry for determining content of 7 conjugated estrogens in livestock and poultry manure
Quantitatively detecting the content of 7 conjugated estrogens enriched from livestock and poultry manure in the sample to be detected in the sample injection bottle obtained by the treatment of the step (2) by adopting an internal standard method and a high performance liquid chromatography tandem mass spectrometer; the 7 conjugated estrogens are respectively: estrone 3- (BETA-D-glucuronide) sodium salt, 17 BETA-estradiol 17-potassium sulfate salt, BETA-estradiol 3-sodium sulfate salt, 17 BETA-estradiol 3- (BETA-D-glucuronide) sodium salt, estriol-3-O-BETA-D-glucuronide sodium salt, estrone 3-sodium sulfate, 17 BETA-estradiol 17-BETA-D-glucuronide;
respectively selecting 17 beta-estradiol D 3 3-BETA-D-glucuronide is used as an internal standard substance of estrone 3- (BETA-D-glucuronide) sodium salt, 17 BETA-estradiol 17-BETA-D-glucuronide and estriol-3-O-BETA-D-glucuronide sodium salt, and 17 BETA-sodium estradiol sulfate-D-glucuronide sodium salt is selected 4 As internal standard substance of estrone 3-sodium sulfate, BETA-estradiol 3-sodium sulfate, and 17 β -estradiol 17-potassium sulfate.
2. The method according to claim 1, wherein in step (1), 100ng of 17 β -estradiol D is added before the microwave extraction 3 3-beta-D-glucuronide, 17 beta-sodium estradiol sulfate-D 4 Both internal standards indicate recovery.
3. The method according to claim 1 or 2, wherein in the step (2), the low-concentration methanol used for washing the solid phase extraction column is methanol-water solution with volume concentration of less than 10%; dissolving the residue with organic solvent, diluting to desired volume, and performing ultrasonic treatment for 5-10 min; filter to a brown injection vial with a needle filter; the needle filter adopts a polytetrafluoroethylene needle filter head with the pore diameter of less than 0.22 mu m.
4. The method according to claim 1 or 2, wherein in the step (2), the organic solvent for dissolving the residual substance after nitrogen blowing is methanol or acetonitrile, or a methanol-water solution with a volume concentration of 70-80%, or an acetonitrile-water solution with a volume concentration of 70-80%.
5. The method according to claim 1 or 2, wherein in the step (3), 7 conjugated estrogens are detected by AB5500Q-trap high performance liquid chromatography tandem mass spectrometer of AB, USA, and Shimadzu 30A liquid chromatography; the liquid chromatography column used was Shim-Pack XR-ODSII C18 column type 75mm × 2mm, 1.0 μm.
6. The method according to claim 1 or 2, wherein, in the step (3), the column temperature of the chromatographic column is 40 ℃; the sample size is 4 mu L; temperature of sample introduction chamber10 ℃; flow rate of mobile phase 0.3 mL/min -1 (ii) a The mobile phase A is an ammonia water-water solution with the volume concentration of 0.1 percent, wherein the water is Milli-Q ultrapure water; the mobile phase B is acetonitrile; the gradient elution procedure was: 0-2min, 10% B; 2.0-2.2min, 10% B-50% B; 2.2-3.5min, 50% B; 3.5-3.8min, 50% B-97% B; 3.8-5.5min, 97% B; 5.5-5.6min, 97% B-10% B; 10min, 10% B.
7. The method according to claim 1 or 2, wherein in step (3), the mass spectrometric detection conditions are: electrospray ion source anion mode ESI-, triple quadrupole mass analyzer, scanning mode: detecting in a multi-reaction monitoring mode MRM; the ion source temperature is 550 ℃, the ionization voltage is-4500V, the air curtain gas CUR is 35psi, the spray gas GS1 is 50psi, the auxiliary heating gas GS2 is 50psi, the collision gas CAD is Medium, and the collision gas is high-purity nitrogen.
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